With the progress of knowledge related to genomics, the need to move from a treatment generally applicable to a given disease to a treatment specifically applicable to a specific individual affected by the such a disease is increasingly felt in medicine. The advantages of a customized therapeutic approach are apparent, where an inadequate selection of patients to be treated leads to a health expenditure which may be contained not only by being able to predict, and thus avoid, often expensive and ineffective treatments for the specific subject but also avoiding adverse patient-specific effects.
In tumor therapy, this need is particularly felt, where the effectiveness of treatments is typically low: even drugs aimed at specific genetic mutations may be ineffective in 80-90% of cases in the absence of an adequate selection of patients (B Majumder et al., Predicting clinical response to anticancer drugs using an ex vivo platform which captures tumour heterogeneity. Nat. Commun. 2015, 6:6169).
With the aim of being able to achieve tests which enable the effective implementation of customized medicine, tests have been successfully developed which are based on the genetic profile and on protein expression (Staunton J E et al, Chemosensitivity predicition by transcriptional profiling. PNAS 2001, 98:10787-10792; van't Veerand L J, Bernarnds R, Enabling personalised cancer medicine through analysis of gene-expression patterns. Nature 2008, 452:564-570). However, especially in tumors, the relevance of the tumor microenvironment in conjunction with other characteristics of the patient to determine the effectiveness of a therapy has been widely demonstrated. To take also these factors into due consideration, the need for ex vivo functional assays is strongly felt (Tan C. et al., Evaluation of a chemoresponse assay as a predictive marker for the treatment of recurrent ovarian cancer: further analysis of a prospective study. British J. Cancer 2014, 111:843-850).
Ex-vivo functional assays are currently available which, where implemented by carrying out the analysis immediately downstream of the biological sample collection and maintaining said sample under controlled conditions and as much as possible similar to those representative of the tumor microenvironment in-vivo, have demonstrated a high ability to predict the efficacy of drug therapies. As an example, a method is described in WO2010/135468. However, the ex-vivo functional assays available to date exhibit important limitations.
In particular, the assays developed for the ex-vivo analysis of the pharmacological activity in hematology and oncology are mainly based on the use of flow cytometry (FACS) and/or fluorimetric or colorimetric assays involving kits used to measure the cell viability and/or proliferation on whole cell populations, such as the metabolic assays MTT, ATP, the MiCK assay (Kravtsov V D & Fabian's Automated monitoring of apoptosis in cell suspension cultures. Lab. Invest. 1996, 74:551-570) and the DiSC assay (Weisenthal L M et al., A novel dye exclusion method for testing in vitro chemosensitivity of human tumors. Cancer Res. 1983, 43:749-757) for measuring ell death and apoptosis. Such assays inevitably have several limitations. Flow cytometry, for example, exhibits the inability to obtain high-content analyses in time-lapse and the inability to work on cell aggregates, as the cellular breakdown is a fundamental prerequisite for running the test, thereby preventing the evaluation of the response of a cell in its context. The techniques which provide a measure on whole populations are generally characterized by the difficulty of carrying out an analysis limited to the tumor subpopulation and are therefore of limited accuracy. In addition, in all the techniques described, the required sample volumes are typically significant and not always compatible with the clinical practice. For example, in order to reduce the invasiveness of the sampling procedures or in the presence of tumors of limited size, such as metastasis, the sample is available in small quantities, for example up to a few thousands cells or a few dozen cells, if the sample comes from a liquid biopsy, i.e. by the isolation of circulating tumor cells, insufficient quantities to be analyzed according to the techniques described. Having a biological sample which comprises between a few thousands cells and a few dozen cells, conducting a cell analysis on such a sample is difficult to implement through the currently existing instruments and, when it can be implemented, the analysis is still limited to one or very few experimental conditions per sample. In even more complex cases in which only a few cells, such as 10-20 cells, are available, no significant data can be obtained using the functional assays currently available. Moreover, functional assays are currently typically carried out by operators who require specialized laboratories, with expertise and equipment which are not easily accessible. The analysis platforms based on flow cytometry, if provided with complete automation, are represented by complex and bulky machinery, therefore hardly adoptable in clinical contexts and, more particularly, in diagnostic laboratories. Likewise, other techniques described require manual performance of the operations and the availability for an entire laboratory to carry out the diagnostic testing. The tests are then carried out in laboratories far from the place of sampling, thereby delaying the start of the test by a few days, often making them not compatible with the timing of clinical practice, which may need the results within 24-48 hours.
WO2012/072822 describes a system with microwells open upwards and downwards, where channels put said microwells in fluidic communication and the geometry of said microwells allows the formation of a meniscus within them on which the cells and/or particles introduced into the same rest. Optionally, said microwells comprise electrodes which allow to control the movements of cells and/or particles into the same microwells.
US2016/161392
The need is strongly felt for a functional assay based on cell analysis capable of giving answers in a short time since obtaining the biological sample, for example within 24-48 hours, and which allows to obtain high-content data on the cells analyzed, i.e. inclusive of morphological information, even in time-lapse, allowing the dynamic analysis of the information detected on the cells in the sample. Moreover, said assay should be as operator-independent as possible and require small volumes of biological sample, so as to also limit the volumes of reagents and drugs to be used in the execution thereof, thus containing costs, maintaining the ability to provide reliable results even with very small biological samples in terms of quantity, such as also having just 20 cells.
The movement of fluids in microfluidic devices typically uses vacuum or pressure pumps and/or valves. The combination of pumps and valves allows a fine control of the movements of fluids in a circuit.
By way of example, Byun et al. (Pumps for Microfluidic Cell Cultures, Electrophoresis 2014, 35:245-257) describe microfluidic devices for cell culture and pumping systems associated therewith. Also Au et al. (Microvalves and micropumps for BioMEM, Micromachines 2011, 2:179-220) describe a wide variety of valves and pumps to be used in specific combinations, each with unique features which make it applicable in certain contexts and not in others.
The available literature shows that there are no standard parameters on which the selection of micro-pumps and micro-valves should be based, thus requiring a specific study for each specific system.
A strongly felt problem is to efficiently manage the bi-directional movement of fluids in a microcircuit, without necessarily having to rely on pumps and valves, which are bulky and demanding from the point of view of purchase and management costs.
A further problem associated with the microfluidic devices based on valves is that, if the integration of valves in the microsystem is contemplated to obtain high parallelism and/or reduced overall dimensions, the technological complexity required is high, for example due to the need of integrating elastomers as well as rigid materials.
The present invention offers a simple and advantageous solution to the problem by allowing the use of a common liquid handling instrument for the high precision charging, pumping and optionally discharging of fluids in a microfluidic device.